Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo

Purpose To assess the relative impact of overexpression of interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN-γ) expressing recombinant herpes simplex virus type 1 (HSV-1) on altering immune responses in ocularly infected mice. Methods BALB/c mice were co-infected ocularly with avirulent HSV-1 strain KOS and avirulent recombinant HSV-1 expressing murine IL-4 (HSV-IL-4). Controls mice were co-infected with KOS + HSV-IL-2 or KOS + HSV-IFNγ. Following ocular infection, virus replication in the eye, corneal scarring (CS), and survival were determined. We also isolated recombinant viruses from eye and trigeminal ganglia of KOS + HSV-IL-4 infected mice. Results In this study we found that ocular infection of BALB/c mice with a mixture of HSV-IL-4 and KOS resulted in increased death and increased eye disease. In contrast, when mice were infected in one eye with KOS and the other eye with HSV-IL-4 no death or eye disease was seen. Intraperitoneal co-infection of mice with KOS and HSV-IL-4 also did not result in HSV-1 induced death. Interestingly, ocular infection of mice with a mixture of HSV-IL-2 and KOS did not have any effect on severity of the disease in infected mice. We isolated recombinant viruses from KOS + HSV-IL-4 infected mice eye and trigeminal ganglia. Some of the isolated viruses were more neurovirulent then either parental virus. Infection of macrophages with IL-4 expressing virus down-regulated IL-12 production by macrophages. Conclusions These results suggest a role for IL-4 in suppression of immune response and generation of virulent viruses in vivo.

Herpes Simplex virus type 1 (HSV-1) is a neurotropic virus that spreads from the site of infection (i.e., eye, genital tract, labial) to the nervous system [1]. In both humans and animal models of HSV-1, virus establishes a latent infection in the ganglia [2]. Based on neurovirulence in animal studies, HSV-1 strains can be classified into two main categories: (1) Avirulent HSV-1 strains, such as strain KOS, do not kill BALB/c mice or New Zealand White (NZW) rabbits following ocular infection; and (2) virulent HSV-1 strains, such as McKrae, that kill ~50% or more BALB/c mice and NZW rabbits following ocular infection [3][4][5][6]. Previously it was shown that footpad infection of mice with a 1:1 mixture of avirulent HSV-1 strains ANG and KOS resulted in a lethal infection in 62% of the infected mice [7,8]. The avirulent phenotype in ANG and KOS appeared to be the result of single amino acid changes to glycoprotein D (gD) or gB, respectively [9,10]. In contrast, to HSV-1 essential genes and the γ34.5 virulence gene [9][10][11], deletion of the latency associated transcript (LAT) does not alter virulence despite reducing reactivation in ocularly infected rabbits and mice [12][13][14]. Using the McKrae derived LAT-deficient virus dLAT2903 [12], we previously constructed recombinant viruses expressing murine IL-2 (HSV-IL-2) and IL-4 (HSV-IL-4), each driven by the LAT promoter [15,16]. These recombinant viruses, in contrast to their parental virus, were avirulent in ocularly infected mice despite having similar replicating kinetics in tissue culture [15,16]. The HSV-IL-2 recombinant virus, but not the HSV-IL-4 recombinant virus, induced central nervous system (CNS) demyelination following ocular infection of mice [17,18]. In this study we set out to determine if co-infection with KOS or HSV-IL-4 would block HSV-IL-2-induced CNS demyelination. Surprisingly, following ocular infection of BALB/c mice with a mixture of KOS and HSV-IL-4, 43% of the infected mice died. We isolated four viruses from trigeminal ganglia and corneas of mice with severe neurologic involvement. These viruses showed a wide range of virulence and corneal scarring. Virulent recombinant viruses were only generated using ocular co-infection of HSV-IL-4 with KOS, and not KOS with HSV-IL-2, HSV-CD80, HSV-IFNγ, HSV-IL-12p35, or HSV-IL-12p40  recombinant viruses.
Ocular infection: Mice were infected ocularly with a mixture of 1×10 5 PFU of KOS plus 1×10 5 PFU of HSV-IL-4, or dbl-IL-4 per eye in 5 μl of tissue culture media as eye drops without prior corneal scarification. Some mice were infected with 2×10 5 PFU/ eye of KOS in one eye and 2×10 5 PFU/ eye of HSV-IL-4 in the other eye. Control mice were infected with 2×10 5 PFU/ eye of KOS, HSV-IL-4, or dbl-IL-4.

Analysis of replication and clearance of HSV-1 from the eye:
Eyes were swabbed once daily on days 1, 3, and 5 post-ocular infection with a Dacron swab (Spectrum type 1). The swab was transferred to a 12×75 mm culture tube containing 1 ml of media, frozen, thawed, and virus titers determined using standard plaque assays on RS cells.

Infection of bone marrow (BM)-derived macrophages in vitro:
Monolayers of macrophages isolated from BALB/c mice were infected with 10 PFU/cell of dLAT2903 (HSV-IL-4 parental virus), HSV-IL-4, or mock-infected. One hour after infection at 37 °C, virus was removed and the infected cells were washed three times with fresh media and fresh media was added to each well. The monolayers including the media were harvested at 12 and 24 h post infection. RNA preparation was done as we previously described [23].
TaqMan Real-Time PCR: The expression levels of IL-12p35 and IL-12p40 genes, along with the expression of the cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (internal control) were evaluated using commercially available TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) with optimized primer and probe concentrations as we previously described [23,24]. Primer-probe sets consisted of two unlabeled PCR primers and the FAM™ dye-labeled TaqMan MGB probe formulated into a single mixture. The primers and probe used were as follows: 1) IL-12p35 (ABI ASSAY I.D. Mm00434165_m1 -Amplicon length=68 bp); 2) IL-12p40 (ABI ASSAY I.D. Mm 01288992_m1 -Amplcon length=109 bp); and 3)IL-4 (ABI Mm00445259_m1 amplicon length=79 bp). GAPDH was used as an internal control (ABI ASSAY I.D. m999999.15_G1 -Amplicon Length=107 bp). The expression level of HSV-1 gB was similarly evaluated using custom made TaqMan Gene Expression Assays (Applied Biosystems). The gB primers and probe were: forward primer, 5′-AAC GCG ACG CAC ATC AAG-3′; reverse primer, 5′-CTG GTA CGC GAT CAG AAA GC-3′; and probe, 5′-FAM-CAG CCG CAG TAC TAC C-3′. Quantitative real-time PCR was performed as we described previously [23]. Real-time PCR was performed in triplicate for each sample from each time point. Relative gene expression levels were normalized to the expression of the GAPDH housekeeping gene (endogenous loading control). Southern analyses: Briefly, viral DNA was digested with BamHI, the restriction fragments were separated in a 0.9% agarose gel, transferred to Zeta paper, rinsed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min, cross-linked to the membrane by UV light, and DNA-DNA hybridization performed with 32 P-labeled IL-4 DNA as previously described [15,25]. Statistical analysis: Fisher's exact tests were performed using the computer program Instat (GraphPad, San Diego, CA) to analyze survival and corneal scarring (CS). Results were considered statistically significant when the "p" value was <0.05. Fisher's exact test). In contrast to the co-infection results, when mice were infected with KOS in the right eye and HSV-IL-4 in the left eye no increase in virulence was observed in infected mice (not shown). In addition, when mice were coinfected with a mixture of KOS and HSV-IL-2 (instead of KOS and HSV-IL-4) no increase in virulence was detected (not shown).

Co-infection of BALB/c mice with avirulent HSV-IL-4 and KOS increases virulence in infected mice
To determine if the increased virulence was associated with IL-4, additional groups of 30 mice (from 4 separate experiments) were co-infected with dbl-IL-4 and KOS. Control mice were infected with dbl-IL-4 alone. One hundred percent of the mice infected with dbl-IL-4 survived the infection at both doses (20/20; Table 2), while 7% (2/30) of mice infected with the dbl-IL-4 + KOS died ( Table 2). Although this difference did not reach statistical significance, it should be noted that the dbl-IL-4 parent virus DM33, is deleted for γ34.5 and LAT, and neither this virus, nor d34.5, deleted for γ34.5, nor KOS, has ever killed a single mouse or rabbit in our hands. Thus, the death of 2 mice with the mixture of dbl-IL-4 + KOS may suggest that this virus mixture was more virulent than either parent. However, we cannot rule out that the death of these 2 mice could be due to other reasons as well. We therefore conclude that mixtures of KOS + a virus expressing IL-4 driven by the LAT promoter resulted in decreased survival (i.e., increased virulence).

Virus replication in mouse tears:
The virus titers in the tear films that had been collected on days 1, 3, and 5 post ocular infection from mice described in Table 1 were determined using plaque assays on RS cells. There were no significant differences among the virus titers in the tear films of mice infected with HSV-IL-4 + KOS compared with mice infected with KOS alone or HSV-IL-4 alone (Figure 1). Similarly no significant differences were detected in mice that were infected in their right eye with KOS compared with the same mice that were infected with HSV-IL-4 on the left eye ( Figure  2). Thus, it appears that there was no direct correlation between acute virus replication in the eye on days 1, 3, or 5 PI and increased virulence in co-infected mice.
Corneal scarring (CS) in surviving mice: CS was measured in all mice that survived until 28 days after ocular infection ( Table 1). The extent of CS was significantly higher in mice co-infected with HSV-IL-4+KOS than mice infected with either HSV-IL-4 or KOS separately (Figure 2; p=0.03 and p<0.0001, respectively). Similarly CS was significantly higher in mice that were co-infected with dbl-IL-4+KOS than mice infected with dbl-IL-4 or KOS separately (Figure 3; p=0.0003). Thus, co-infection of mice with KOS and two different recombinant viruses expressing IL-4, increased severity of CS in surviving mice.
Virulence and CS with viruses isolated from eyes and trigeminal ganglia of co-infected mice: HSV-1 was isolated from eyes and TGs of mice co-infected with HSV-IL-4 + KOS, following euthanasia on day 6 post infection. Tissues were ground up and total supernatants were grown on RS cells   BALB/c mice were infected ocularly with 2×10 5 PFU/eye of each virus isolated from eye or TG of mice following co-infection with HSV-IL-4+KOS mixtures described in Table 1. Survival was determined 28 days post infection as described in the Methods.
as described in the Methods. Viral supernatants were plaque purified and after three cycles of plaque purification, four of the plaque purified viruses isolated from eyes and TGs were used for further study. Groups of 20 BALB/c mice were infected ocularly with 2×10 5 PFU/eye of each of the 4 plaque purified viruses (i.e., vEye2, vTG2, vEye3, or vTG3). All mice (100%) infected with vEye2 or vTG2 virus survived ocular infection (Table 2). In contrast, only 80% (16/20) and 20% (4/20) of mice infected with vEye3 and vTG3 survived ocular infection, respectively. This difference between mice infected with vEye3 compared with mice infected with each individual virus was highly significant (p=0.0001, Fisher's exact test). CS was measured in surviving mice shown in Table 2. The Level of CS for mice infected with vEy2, vTG2, and vTG3 was the same as mice co-infected with HSV-IL-4+KOS (Figure 4; p>0.05). However, CS in mice that were infected with vEye3 virus was significantly higher than other groups or co-infected mice described in Table 1 (Figure 4; p<0.001). Thus, as a result of co-infection we have isolated a virus that is more pathogenic than either individual parental virus or coinfection with a mixture of both parental viruses.

Structure of isolated viruses:
HSV-IL-4 was derived from the dLAT2903 strain by the insertion of the IL-4 gene and restoration of the LAT promoter so that the inserted IL-4 gene is under control of the endogenous LAT promoter [15]. To determine if vEye2, vEye3, vTG2, and vTG3 still contain the IL-4 insert, the genomic structure of each virus was confirmed by restriction enzyme analysis, and Southern blot ( Figure 5). Similar to HSV-IL-4, the vEye2, vEye3, vTG2, and vTG3 viruses all had the IL-4 insert. The size of the IL-4 insert was similar to that of IL-4 from pLAT-IL-4 ( Figure 5). As expected KOS DNA was negative for presence of IL-4 ( Figure 5). Thus, the size of the IL-4 gene in the isolated recombinant viruses was similar to the IL-4 gene in the parental HSV-IL-4 virus.
To confirm that the LAT promoter was functional in the isolated viruses, confluent monolayers of RS cells were infected at a multiplicity of 10 PFU/cell of HSV-IL-4, vEye2, vEye3, vTG2, or vTG3. Infected cells were collected 24 and 48 h post infection and total RNA was isolated for detection of the IL-4 transcript by TaqMan RT-PCR as described in the Methods. At 48 h post infection, the levels of IL-4 transcript were similar for all viruses, except vTG3, which appeared higher ( Figure 6A; 48 h). This suggested that the increased neurovirulence of vEye3 was not due to decreased expression  Figure 6A; 24 h). Thus, it is possible, but we think unlikely, that reduced IL-4 expression early in infection could be involved with increased neurovirulence of vEye3. HSV gB transcript levels were examined as a control ( Figure 6B). The gB RNA levels followed the same patterns seen for IL-4 RNA, except for vEye2 which had gB RNA levels similar to the parental virus at 24 h post infection. Similar patterns of IL-4 RNA levels were detected when RS cells were infected for 12 h or 24 h with 1PFU/cell of each virus (not shown). To confirm that the IL-4 transcripts were being translated into protein, the media from the infected RS cells described above were subjected to ELISA as we described previously [20]. All four viruses appeared to express similar levels of IL-4 (not shown). Together, these results suggest that the observed increased virulence detected with the isolated recombinant virus vEYe2 was not due to reduced expression of IL-4 compared to the parental HSV-IL-4 virus.

Down-regulation of IL-12p35 and IL-12p40 transcripts in BM-derived macrophages infected with HSV-IL-4: Since IL-4
is an indicator of TH2 response and macrophages play a major role in pushing the immune response toward TH1 and away from TH2 by IL-12 production, we investigated the possibility of whether HSV-IL-4 suppresses IL-12p35 and IL-12p40 transcripts. Macrophages were isolated from BALB/c mice and infected with 10 PFU/cell of HSV-IL-4, dLAT2903, or mock infected. Infected or mock-infected macrophages were harvested 12 and 24 h post infection and total RNA was isolated as described in Materials and Methods. The levels of IL-12p35 and IL-12p40 mRNAs were quantitated by TaqMan RT-PCR. Cellular GAPDH mRNA was used as an internal control. Our results suggest that compared to dLAT2903, HSV-IL-4 suppressed expression of both IL-12-35 ( Figure  7A) and IL-12p40 transcripts ( Figure 7B). The pattern of IL-12p35 and IL-12p40 transcript in KOS infected macrophages were similar to that of dLAT2903 (not shown). These results suggest that HSV-IL-4 infection suppresses IL-12 responses in infected macrophages and this may skew the TH1 response toward a TH2 response.

DISCUSSION
IL-4 has a broad range of biologic and immunological activities [26,27] and is considered an indicator of a TH2 response [27][28][29]. IL-4 is secreted by activated CD4 + TH2 cells [30], CD8 + TC2 cells [31], mast cells [32], and basophils [33,34]. In this study, we have shown that ocular infection of mice with a mixture of two avirulent HSV-1 viruses, in which one of the viruses expresses murine IL-4 increased viral pathogenesis. In contrast, when we co-infected mice with recombinant viruses expressing other cytokine genes and HSV-1 strain KOS no increase of pathogenesis and neurovirulence was detected in infected mice. Our coinfection result is similar to mousepox virus expressing IL-4 which has increased virulence [35]. This may be because the mousepox virus expressing IL-4 resulted in reduced IFN-γ gene expression [35]. Figure 3. Corneal scarring in co-infected mice. Corneal scarring in surviving mice described in Table 1   Corneal scarring in surviving mice described in Table 2 was examined on day 30 PI as described in the Methods. CS score represents the average±SEM from 40, 40, 8, and 32 eyes infected with vEY2, vTG2, vEye3, or vTG3, respectively.
Although IL-4 enhances TH2 development [27,28], however the effect of IL-4 expressed by recombinant HSV-1 on TH1 responses may not be a direct effect. Our results suggest that IL-4 has a suppressive effect on IL-12 expression, while previously it was shown that exogenous application of IL-4 is upregulating the production of IL-12 [36]. This discrepancy could be due to use of a recombinant virus expressing IL-4 rather then adding rIL-4 to the culture. Interleukin-12 (IL-12) is a pleiotropic heterodimeric glycoprotein composed of a 35-kDa α subunit and a 40-kDa β subunit [37,38]. The IL-12 heterodimer may bias the response in favor of the production of TH1 cells through its ability to drive the differentiation of TH0 cells into TH1 cells [39][40][41]. Thus, our results may suggest that IL-4 suppression of IL-12 may bias the TH1 response toward a TH2 response and this may lead to increase of recombination in vivo. In line with this finding, previously we have shown that HSV-1 replicated to higher titers in the eyes of IL-2 −/− mice which have higher TH2 response then WT or IL-4 −/− mice [42]. Furthermore, we have reported that in IL-4 −/− mice, which are deficient in IL-4 production, lack a TH2 response, and have elevated IL-2 response, HSV-1 replicated to lower titers and ocular HSV-1 replication could be increased by exogenously added rIL-4 [42]. Previous studies also have shown that delayed viral clearance was seen in mice challenged with influenza virus in the presence of exogenously applied IL-4 [43], following respiratory syncytial virus infection of transgenic mice expressing IL-4 [44], and following infection of mice with a vaccinia virus recombinant expressing IL-4 [45]. Thus, the present study suggests that IL-4 expressed by  Total RNA was isolated 24 and 48hr post infection and TaqMan RT-PCR was performed using IL-4-and gBspecific primers as described in the Methods. In each experiment, an estimated relative copy number of IL-4 and gB were calculated using standard curves generated from pVR1055-IL-4 and pVR1055-gB, respectively. Briefly, DNA template was serially diluted 10fold such that 5 μl contained from 10 3 to 10 11 copies of IL-4 or gB, then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH was used as internal control. Each point represents the mean±SEM (n=4). Panel A indicated gB and panel B indicates IL-4.
HSV-1 increases virus recombination by shifting the immune response from a TH1 to a TH2. Similar to this study, in another study, IL-4 expression by a recombinant vaccinia virus exacerbated infection and the IL-4-induced exacerbation was T cell independent [46].
In summary, co-infection of two avirulent HSV-1 in which one of the two viruses expressing IL-4 generated recombinant viruses in vivo. These recombinant viruses were more pathogenic and more virulent then their parental viruses. Infection of macrophages with IL-4 expressing virus downregulated IL-12 production by macrophages. These findings suggest a role for IL-4 in suppression of immune response and generation of virulent viruses in vivo. Figure 7. Level of IL-12p35 and IL-12p40 transcripts in macrophages infected with HSV-IL-4. Subconfluent monolayers of macrophages were infected with 10 PFU/cell of HSV-IL-4 or parental virus. Total RNA was isolated 12 and 24 h post infection and TaqMan RT-PCR was performed using IL-12p35-and IL-12p40-specific primers as described in the Methods. IL-12p35 and IL-12p40 mRNA levels were normalized in comparison to each transcript in mock-infected cells. GAPDH was used as internal control. Each point represents the mean±SEM (n=8).